Playing the game is divided between finding a room to play in, selecting a song, and playing a song. There are Music Halls, technically servers, on which a user can play in. Some music halls are named after the muses in Greek mythology.

After choosing a server on which to play, the player (commonly called an O2Jammer in the O2Jam community) is presented with a list of "channels," each of which allows a maximum of 100 users at a time. Upon selecting a channel, the player is presented with a list of rooms. The types of rooms on O2Jam are Single rooms, VS rooms, Album rooms, Couple rooms, and Jam rooms (not implemented).

Over 350 songs on the Filipino server and 270 on the Malaysian server are currently available to play Including all O2Jam versions, there are over 580 songs available for playing.

The genres that the songs are part of run the gamut from piano pieces to techno versions of classical pieces to songs that are reminiscent of J-Pop, with an unusually large number of Christmas -themed songs. Most of the songs have 3 difficulty levels of play. The harder the difficulty, the more notes, drum beats, and other sounds need to be played by the player, as opposed to automatically being played in the background. On the hardest difficulty, many songs have a limited amount of music not being played by the user.

To be able to play a song in a VS Room, the song must either be free and each player have the song downloaded, or be paid for and downloaded by each player. To play in an Album Room, all songs in the album being played must have been downloaded before the album can be played.

A small optional step while selecting the song is selecting the background that all of the players will play on. This is mostly a cosmetic change, since all this does is determine what playing field the avatars will be standing on and what font the combo counter and note quality indicator (Cool/Good/etc.) will be in, as well as determining the visual effect that displays whenever a note is hit well. By default, the background is random.

Horizontal bars slide down a series of seven columns, each corresponding to a key on the keyboard. Just before the notes hit the bottom of the column, the player has to hit the key corresponding to that column. Each 'key press' emits a note, vocal, or other instrument that creates a full-sounding song. A correct hit is also rewarded with a small 3D visual effect that is determined by the background on which the players are playing.

The grading scale used by the game is, from most accurate press to least, Cool, Good, Bad, and Miss. Consecutive Cool and Good notes add both to the player's combo and to their Jam meter. When the Jam meter fills up (if you get 25 Cools, 50 Goods, or a combination all in one streak), a large number flashes in front of the player's avatar indicating their current Jam Combo. The goal of the player is to get both a high individual note combo as well as a high Jam Combo. Because the Jam meter fills up faster when a player hits a Cool instead of a Good, two players can both get a full combo (hitting every note as a Cool or Good) but still have a different Jam Combo.

When the player gets fifteen Cools in a row, a blue pill appears in the pill meter, with a maximum of five pills. Each one of these pills will transform a Bad hit into a Cool, keeping the player's combo going.

After all of the players are done playing the song, their individual scores are ranked against each other, and all players are awarded Gems based on how well they performed.

One way players challenge themselves is by increasing or decreasing the speed at which the notes descend. The multipliers from the "base speed" are x0.5, x1, x1.5, x2, x2.5, x3, x4, x5, x6, and x8. These modifiers are often points of contention in VS Rooms, as many players try to play a song at the fastest speed possible, while others prefer to have the notes a bit slower. With many harder songs, x1 is used to increase the challenge, as the notes end up closer together, making it harder to read. Speeding up the notes does not affect the music at all, but instead spreads out the notes, making them (in non-extreme cases) easier to read in relation to each other, though the notes appear on screen for less time.